Most human tissues undergo homeostatic self-renewal wherein aged, damaged cells are replaced by the offspring of resident stem cells. The systems that orchestrate this regenerative growth operate with astounding fidelity, but over the long term even minor dysfunctions in tissue homeostasis can cause problems ranging from atrophy and organ failure to chronic inflammation and cancer. Despite being critical for health and longevity, tissue homeostasis remains poorly understood. This project addresses mechanisms of epithelial renewal in the intestine (midgut) of Drosophila. The cell types and signaling interactions in the fly's midgut are similar to those in the human and mouse intestine, but its superlative genetic tools allow rapid, incisive experimentation. We've outlined a feedback system explaining gut epithelial renewal in Drosophila that should be highly relevant to endodermal human epithelia of the GI, respiratory, renal, and urinary systems. When the fly's intestinal enterocytes (ECs) are damaged, the gut produces IL-6 like cytokines (Upd2, Upd3) that trigger inter-cellular EGFR signaling, which in turn activates intestinal stem cells (ISCs) to produce new ECs. We address the least well-understood aspects of this feedback mechanism in three specific aims. In Aim 1 we test candidate pathways (Jnk, p38, Hpo, ROS), and do unbiased RNAi screening to map the damage response network and identify the upstream sensors that initially trigger the response. A better understanding of epithelial damage sensing could improve cancer and inflammation therapy, and fuel advances in regenerative medicine. Aim 2 delves into the most poorly understood aspect of EGFR action, namely how downstream MAPK effectors control cell growth and division. Combining genome-wide chromatin profiling, binding site mapping of essential MAPK-dependent transcription factors, and mRNA expression profiling, we generate high resolution genomic maps of how MAPK signaling controls gene expression to activate the ISC cell cycle. We also test whether EGFR's transcriptional outputs drive cell growth, and if this is not the case, we'll perform a phospho-proteomics screen to identify the MAPK effectors that do. The results could lead to better diagnostics and alternative therapies for prevalent EGFR/RAS/RAF-driven inflammatory diseases (e.g. IBD) and cancers (colorectal, breast, pancreatic, skin, lung, glial). Aim 3 leverages our in vivo data to develop in vitro culture of Drosophila ISCs and gut organoids. This will enable many new approaches for studying stem cell biology using this genetically powerful system, including live imaging, biochemistry and high throughput screening. In sum this project will illuminate two key mechanisms of tissue homeostasis in an endodermal epithelium: how epithelia sense stress, and how resident stem cells respond by growing and proliferating. New gene functions and signaling connections will be defined, highlighting novel strategies for cancer and inflammation therapy and regenerative medicine.